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PD-L1-mediated gasdermin C expression switches apoptosis to pyroptosis in cancer cells and facilitates tumour necrosis

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An Author Correction to this article was published on 08 October 2020

This article has been updated

Abstract

Although pyroptosis is critical for macrophages against pathogen infection, its role and mechanism in cancer cells remains unclear. PD-L1 has been detected in the nucleus, with unknown function. Here we show that PD-L1 switches TNFα-induced apoptosis to pyroptosis in cancer cells, resulting in tumour necrosis. Under hypoxia, p-Stat3 physically interacts with PD-L1 and facilitates its nuclear translocation, enhancing the transcription of the gasdermin C (GSDMC) gene. GSDMC is specifically cleaved by caspase-8 with TNFα treatment, generating a GSDMC N-terminal domain that forms pores on the cell membrane and induces pyroptosis. Nuclear PD-L1, caspase-8 and GSDMC are required for macrophage-derived TNFα-induced tumour necrosis in vivo. Moreover, high expression of GSDMC correlates with poor survival. Antibiotic chemotherapy drugs induce pyroptosis in breast cancer. These findings identify a non-immune checkpoint function of PD-L1 and provide an unexpected concept that GSDMC/caspase-8 mediates a non-canonical pyroptosis pathway in cancer cells, causing tumour necrosis.

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Fig. 1: PD-L1 translocates into the nucleus in response to hypoxia.
Fig. 2: p-Y705-Stat3 binds to PD-L1 and facilitates its nuclear translocation under hypoxia.
Fig. 3: nPD-L1 switches TNFα-induced apoptosis to pyroptosis under hypoxia.
Fig. 4: nPD-L1 and p-Y705-Stat3 act as co-activators in the transcriptional activation of GSDMC expression in response to hypoxia.
Fig. 5: GSDMC cleavage by caspase-8 determines the hypoxia-induced apoptosis-to-pyroptosis switch with TNFα treatment.
Fig. 6: The N-terminal domain of GSDMC (amino acids 1–365) binds to the cell membrane and induces pyroptosis.
Fig. 7: GSDMC, nPD-L1 and caspase-8 are required for TNFα-induced tumour necrosis in hypoxic regions.
Fig. 8: Chemotherapeutic drugs induce pyroptotic cell death via the nPD-L1/GSDMC-mediated non-canonical pathway in breast tumours.

Data availability

The consensus caspase-8 recognition motifs were analysed using the CaspDB database (http://caspdb.sanfordburnham.org/). The p-Y705-Stat3-binding site in the GSDMC promoter was analysed using the GPMiner program (http://gpminer.mbc.nctu.edu.tw/). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Change history

  • 08 October 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank K. Dunner Jr. and acknowledge the National Institutes of Health Cancer Center (Support grant no. P30CA016672) for supporting the High Resolution Electron Microscopy Facility at The University of Texas MD Anderson Cancer Center for the electron microscopy analysis. This work was funded in part by the National Institutes of Health (grant nos P30CA016672 and R01 AI116722 (to M.-C.H.)); a National Breast Cancer Foundation Breast Cancer Research Foundation grant to M.-C.H.; the Patel Memorial Breast Cancer Endowment Fund; the Sister Institution Network Fund for collaboration between The University of Texas MD Anderson Cancer Center and China Medical University (to M.-C.H.); the YingTsai Young Scholar Award (grant no. CMU108-YTY02 to J.-M.H.); China Medical Univesity Start Up Fund (grant no. CMU108-N-02 to W.-J.W.); the NIH (grant nos R35 CA220430 and PO1 CA092584); the Cancer Prevention and Research Institute of Texas (grant no. RP180813); Robert A. Welch Chemistry Chair (to J.A.T.); Cancer Prevention and Research Institute of Texas (grant no. RP160710 to M.-C.H.); and Drug Development Center, Ministry of Education, Taiwan (grant no. 108-2311-B-241-001 to M.-C.H. and Y.C.).

Author information

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Authors

Contributions

J.H. and M.-C.H. designed and conceived the study. J.H. and M.-C.H. wrote the manuscript. J.L.H. and K.H. contributed to the preparation of the manuscript. J.H., R.Z., W.X., Y. You, J.-M.H., Y.C., Y.-C.W., C.L., W.-J.W., B.K., Z.Y., Y. Yang, X.X., Y.L., C.-W.L., B.S. and J.A.T. performed experiments and analysed data. L.N. and C.-W.C. analysed the PD-L1 and GSDMC sequences. Y.W. provided patient tissue samples. M.-C.H. supervised the entire project.

Corresponding author

Correspondence to Mien-Chie Hung.

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Extended data

Extended Data Fig. 1 Establishment of stable cells in the MDA-MB-231 cell line.

(a) Deletion of endogenous Stat3 by CRISPR–Cas9. KO, knockout. Blots are representative of three independent experiments. (b) Stable re-expression of Stat3-WT or Stat3-Y705F in Stat3-knockout MDA-MB-231 cells. Validation of p-Y705-Stat3 expression level by immunoblotting. Blots are representative of three independent experiments. (c) Analysis of the nuclear localization signal (NLS) and nuclear export signal (NES) in the human PD-L1 amino acid sequence compared with that of other species. Sequence alignment of PD-L1 from human, mouse, cow, rat, and dog. The alignment was generated and presented by the ClustalW2 algorithm and ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Identical residues are indicated by the dark red background and conserved residues are in red. The NLS and NES sequences of PD-L1 are conserved across species. (d-f) Stable re-expression of PD-L1-WT (wild-type PD-L1), PD-L1-NLS (NLS-mutated PD-L1) or PD-L1-NES (NES-mutated PD-L1) in the PD-L1–knockout MDA-MB-231 cell line. Immunoblotting of total PD-L1 expression level in stable transfectants (d). Blots are representative of three independent experiments. Representative images of PD-L1 distribution of three independent experiments was determined by confocal assay under hypoxia (e-f). Scale bar, 20 μm. Unprocessed blots are provided in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 nPD-L1-mediated pyroptotic cell death in cell lines of multiple cancer types.

(a) Immunoblotting analysis of PD-L1 expression levels in liver cancer and lung cancer cell lines. Blots are representative of three independent experiments. (b) Hypoxia-induced pyroptotic cell death in PD-L1-positive cells of multiple cancer types treated with p-Stat3 inhibitor, HO-3867. Red arrows indicate the pyroptotic cells with big bubbles. The experiment was repeated three times with similar results. Scale bar, 20 μm. Unprocessed blots are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 nPD-L1 and p-Y705-Stat3 are required for hypoxia-induced GSDMC expression.

(a) Real-time quantitative polymerase chain reaction (RT–qPCR) analysis of mRNA levels of gasdermin family members in the indicated MDA-MB-231 stable transfectants. Data are shown as mean ± SD of n = 3 independent experiments. (b and c) GSDMC mRNA (b) and protein (c) levels in MDA-MB-231 cells under hypoxia. Data of (b) are shown as mean ± SD of n = 3 independent experiments. Blots are representative of three independent experiments. N, normoxia; H, hypoxia. (d) RT–qPCR analysis of mRNA levels of GSDMC in the indicated MDA-MB-231 stable transfectants. Data are shown as mean ± SD of n = 3 independent experiments. (e-g) MDA-MB-231 cells were cultured under hypoxia and treated with HO-3867 (20 μM) or ivermectin (25 μM). GSDMC mRNA (e) and protein (f) levels were analysed by RT–qPCR and immunoblotting, respectively. GSDMC promoter luciferase reporter plasmids were transfected to cells, and luciferase activity was measured in (g). Data of (e) and (g) are shown as mean ± SD of n = 3 independent experiments. Blots are representative of three independent experiments. P values of all statistical analysis were determined by two-sided Student’s t-test. Statistical source data and unprocessed blots are provided in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 The p-Y705-Stat3 binding site (in red) in the nucleic acid sequence of GSDMC promoter and the expression level of GSDME in MDA-MB-231 cells.

(a) GSDMC promoter sequence was obtained from GeneCopoeia and analysed using the GPMiner program (http://gpminer.mbc.nctu.edu.tw/). Two putative p-Y705-Stat3-binding sites in the GSDMC promoter were predicted. p-Y705-Stat3-binding site was validated by screening with luciferase reporter assay. (b-d) Immunoblotting of GSDME in MDA-MB-231 cells. H226 cells as a positive control. Immunoblotting of the endogenous GSDME (b). Immunoblotting of GSDME in MDA-MB-231 cells under hypoxia (c). Immunoblotting of GSDME in MDA-MB-231 cells treated with daunorubicin, doxorubicin, epirubicin, and actinomycin D (d). The experiments of (b-d) were repeated three times with similar results respectively. Unprocessed blots are provided in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 Caspase-8, but not caspase-6, is required for TNFα-induced pyroptosis under hypoxia.

(a and b) MDA-MB-231 cells were cultured under normoxia (N) or hypoxia (H) and treated as indicated. LDH-released cell death was measured as shown in (a). Data are shown as mean ± SD of n = 3 independent experiments. P values were determined by two-sided Student’s t-test. Representative images of dying cell morphology of three independent experiments (b). Red arrows indicate cell swelling with big bubbles. Scale bar, 20 μm. Caspase-3i, caspase-3 inhibitor Z-DEVD-FMK; Caspase-6i, caspase-6 inhibitor Z-VEID-FMK; Caspase-8i, caspase-8 inhibitor Z-IETD-FMK (each at 10 μM). Statistical source data are provided in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 The pyroptotic cell death in cell lines with different GSDMC and PD-L1 expression status.

(a) Immunoblotting of PD-L1 and GSDMC in Hs578T, BT549, MDA-MB-231 and MCF-7 cells. Blots are representative of three independent experiments. (b) LDH-released cell death in Hs578T, BT549, MDA-MB-231 and MCF-7 cells treated with TNFα under normoxia or hypoxia. Data are shown as mean ± SD of n = 3 independent experiments. P values were determined by two-sided Student’s t-test. Statistical source data and unprocessed blots are provided in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 The N-terminal domain (1–365) of GSDMC is sufficient to induce pyroptosis.

(a) 3D structural modelling of GSDMC. The modelling was performed using SWISS-MODEL. The structural fold of GSDMC N- and C-terminal domains is in blue cartoon presentation. The linker region and the solvent-exposed loop region of the C-terminal domain of GSDMC are coloured in green with numbered Asp/Glu residues coloured in magenta. The structural figure was prepared using PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. (b) Confocal microscopy analysis of the distribution of GSDMC-FL, GSDMC (1¬–365), and GSDMD-N expressions in HeLa cells. GSDMD-N, N-terminal domain of GSDMD. Images are representative of three independent experiments. Scale bar, 20 μm. (c–d) HeLa cells were transfected with GSDMC-FL or GSDMC (1–365). Representative images of dying cell morphology of three independent experiments (c). Scale bar, 20 μm. Dying cells were stained with nucleic acid dye SYTOX green. Red arrows indicate cell swelling with large bubbles. LDH-released cell death is shown as mean ± SD of n = 3 independent experiments (d). P values were determined by two-sided Student’s t-test. (e) HEK293T cells were transfected with Flag-GSDMC-FL or Flag-GSDMC (1–365) for 16 h. Supernatants and cell pellets were collected and subjected to immunoblotting. The blot is representative of three independent experiments. (f) Cartoon diagram of GSDMC structure and the cleavage by caspase-8 across species. Statistical source data and unprocessed blots are provided in Source Data Extended Data Fig. 7.

Source data

Extended Data Fig. 8 Nuclear PD-L1 translocation in vivo causes poor survival in both immunocompetent and nude mice.

(a) IHC staining of HIF1α, GSDMC, PD-L1, and p-Y705-Stat3 in MDA-MB-231 xenografts in nude mice (n = 8). MDA-MB-231 cells were injected subcutaneously into nude mice. Four weeks after tumour cell injection, all mice were sacrificed and tumours excised for IHC staining. The expression levels and patterns of HIF1α, GSDMC, PD-L1, and p-Y705-Stat3 in normoxic and hypoxic area in tumours were analysed and indicated by green arrows. Scale bar, 100 μm. The experiment was repeated three times with similar results. (b) Survival analysis of 4T1 cells expressing PD-L1-WT or PD-L1-NLS in BALB/c nude mice. Data are shown of n = 10 mice. The experiment was repeated three times with similar results. Statistical significance for survival analysis was determined using a log-rank (Mantel–Cox) test. (c) Survival analysis of 4T1 cells expressing PD-L1-WT or PD-L1-NLS in immunocompetent BALB/c mice. Data are shown of n = 10 mice. The experiment was repeated three times with similar results. Statistical significance for survival analysis was determined using a log-rank (Mantel–Cox) test. Statistical source data are provided in Source Data Extended Data Fig. 8.

Source data

Extended Data Fig. 9 Chemotherapeutic drugs induce pyroptotic cell death by nPD-L1/GSDMC-mediated non-canonical pathway in breast tumour.

(a) Representative images of dying cell morphology by phase-contrast microscopy in MDA-MB-231 stable transfectants as indicated treated with daunorubicin, doxorubicin, epirubicin, and actinomycin D. The experiment was repeated three times with similar results. Scale bar, 20 μm. (b) LDH-released cell death in MDA-MB-231 stable transfectants as indicated treated with daunorubicin, doxorubicin, epirubicin, and actinomycin D. Data are shown as mean ± SD of n = 3 independent experiments. P values were determined by two-sided Student’s t-test. Statistical source data are provided in Source Data Extended Data Fig. 9.

Source data

Extended Data Fig. 10 A proposed model of nuclear PD-L1-mediated apoptosis-to-pyroptosis switch under hypoxia.

Under normoxia, caspase-3 can be activated by TNFα-induced caspase-8 to cause apoptotic cell death. However, hypoxia-activated p-Stat3 facilitates nPD-L1 translocation. nPD-L1 cooperates with p-Stat3 to transcriptionally activate GSDMC expression. GSDMC is specifically cleaved by macrophage-derived TNFα-activated caspase-8, which generates an N-terminal pore-forming fragment that causes pyroptotic cell death to induce tumour necrosis in breast cancer.

Supplementary information

Reporting Summary

Supplementary Video 1

MDA-MB-231–GFP cells were treated with TNFα plus CHX under normoxia. MDA-MB-231–GFP, MDA-MB-231 cells with stable expression of GFP protein.

Supplementary Video 2

MDA-MB-231–GFP cells were treated with TNFα plus CHX under hypoxia.

Supplementary Video 3

MDA-MB-231–GFP cells were co-treated with TNFα plus CHX and the Stat3 inhibitor HO-3867 (20 μM) under hypoxia.

Supplementary Video 4

MDA-MB-231–GFP cells were co-treated with TNFα plus CHX and caspase-8 inhibitor Z-IETD-FMK (10 μM) under hypoxia.

Supplementary Table 1

The final concentrations for TNFα, CHX and various stimuli.

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Hou, J., Zhao, R., Xia, W. et al. PD-L1-mediated gasdermin C expression switches apoptosis to pyroptosis in cancer cells and facilitates tumour necrosis. Nat Cell Biol 22, 1264–1275 (2020). https://doi.org/10.1038/s41556-020-0575-z

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